Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses

Abstract

Colletotrichum species are fungal pathogens that devastate crop plants worldwide. Host infection involves the differentiation of specialized cell types that are associated with penetration, growth inside living host cells (biotrophy) and tissue destruction (necrotrophy). We report here genome and transcriptome analyses of Colletotrichum higginsianum infecting Arabidopsis thaliana and Colletotrichum graminicola infecting maize. Comparative genomics showed that both fungi have large sets of pathogenicity-related genes, but families of genes encoding secreted effectors, pectin-degrading enzymes, secondary metabolism enzymes, transporters and peptidases are expanded in C. higginsianum. Genome-wide expression profiling revealed that these genes are transcribed in successive waves that are linked to pathogenic transitions: effectors and secondary metabolism enzymes are induced before penetration and during biotrophy, whereas most hydrolases and transporters are upregulated later, at the switch to necrotrophy. Our findings show that preinvasion perception of plant-derived signals substantially reprograms fungal gene expression and indicate previously unknown functions for particular fungal cell types.

Figure 1. Phylogeny and infection of the two Colletotrichum species analyzed in this study.

(a) Cladogram showing the phylogenetic relationship of Colletotrichum to other sequenced fungi, including 13 species used for comparative analyses (see Fig. 3). The unscaled tree was constructed using CVTree with Rhizopus oryzae as the outgroup. (b) Infection process of C. higginsianum (Ch) and leaf anthracnose symptoms on Brassica and Arabidopsis. The Brassica image is reproduced with permission of University of Georgia Plant Pathology Archive (bugwood.org/). (c) Infection process of C. graminicola (Cg), and leaf-blight, top die-back and stalk-rot symptoms on maize. SP, spore; AP, appressorium; PH, biotrophic primary hyphae; SH, necrotrophic secondary hyphae.

Figure 2. Conservation of synteny between the genomes of C. graminicola and C. higginsianum.

(a) Dot plot showing the syntenic blocks between the 13 chromosomes (optical linkage groups) of C. graminicola (horizontal axis) and the 12 chromosomes of C. higginsianum (vertical axis). Homologies between chromosomes of each species are highlighted in red dashed boxes. Homologous sequences of C. graminicola chromosome 9, indicated between the blue dashed lines, are dispersed among many C. higginsianum chromosomes. (b) Global view of syntenic alignments between the genomes of C. graminicola and C. higginsianum. Linkage groups of C. graminicola are shown as the reference, with linkage group lengths defined by the C. graminicola optical map. For each chromosome, numbered genomic scaffolds (dark gray) positioned on the optical linkage groups are separated by scaffold breaks. The magenta blocks show syntenic mapping of the C. higginsianum sequences; notably, there is a near absence of homologous sequences among the minichromosomes.

Figure 3. Comparison of fungal carbohydrate-active enzyme (CAZyme) repertoires.

(a) Hierarchical clustering of CAZyme classes from Colletotrichum and 13 other fungal genomes. GH, glycoside hydrolase; GT, glycosyltransferase; PL, polysaccharide lyase; CE, carbohydrate esterase; CBM, carbohydrate-binding module. The numbers of enzyme modules in each genome are shown. Overrepresented (orange to red) and underrepresented modules (pale yellow to white) are depicted as fold changes relative to the class mean. (b) Comparison of the pectin-degrading enzyme repertoires of C. higginsianum and C. graminicola shown as the number of modules in each CAZyme family. In total, C. higginsianum encodes 86 such modules, whereas C. graminicola encodes only 42.

Figure 4. Structure and transcription of a secondary metabolism gene cluster.

(a) Gene cluster 18 from C. graminicola (Cg) is orthologous to cluster 10 from C. higginsianum (Ch). The latter is split between four small scaffolds in the Broad Institute genome annotation (supercontigs (SCs) 37, 481, 2,277 and 4,474) and was reconstructed based on an improved genome assembly (Supplementary Note). Microsynteny is indicated by gray bars. The 14 genes highlighted in red in the C. higginsianum cluster are co-regulated. Functional annotation for the cluster genes is provided in Supplementary Table 12. (b) Visualization of RNA-Seq coverage across the C. higginsianum polyketide biosynthesis cluster. The gray curves indicate read coverage (log scale) for the four samples. Co-regulated gene models are highlighted in red. VA, in vitro appressoria; PA, in planta appressoria; BP, biotrophic phase; NP, necrotrophic phase.

Figure 5. Expression profiling of pathogenicity-related genes in C. higginsianum.

(a) Schematic representation of the four C. higginsianum developmental stages selected for RNA sequencing. Gray indicates polystyrene, green indicates living plant cell, and brown indicates dead plant cell. Hpi, hours post-inoculation. (b) Heatmaps of gene expression showing the 100 most highly expressed and significantly regulated genes (log2 fold change >2, P < 0.05) in five functional categories. Overrepresented (pale red to dark red) and underrepresented transcripts (pale blue to dark blue) are shown as log2 fold changes relative to the mean expression measured across all four stages. The arrow indicates the CSEP-encoding gene ChEC6 (CH063_01084). (c) The statistical significance of gene induction (y axis) in five functional categories during fungal developmental transitions (x axis). The P values were calculated using a one-sided Fisher's exact test and represent the probability of observing the number of significantly induced genes for a specific category during a transition given the total number of significantly induced genes during that transition (log2 fold change >2, P < 0.05) and the total number of genes in the category. (d) Transcriptional regulation of the effector gene ChEC6 by plant-derived signals. Confocal micrographs showing C. higginsianum expressing the mCherry reporter gene under the native ChEC6 promoter (overlays of bright-field and fluorescence channels). Appressoria (A) formed on polystyrene are unlabeled (top left), whereas those on the leaf surface (top right) have fluorescent cytoplasm. After host penetration, labeling is visible in young biotrophic hyphae (YH) but not older biotrophic hyphae (OH) (bottom). Scale bars, 10 μm. C, conidium.